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| 2. |
- Hägerhäll, Cecilia, et al.
(författare)
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Transmembrane topology and axial ligands to hemes in the cytochrome b subunit of Bacillus subtilis succinate:menaquinone reductase
- 1995
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 34:35, s. 11080-11089
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Tidskriftsartikel (refereegranskat)abstract
- The membrane-anchoring subunit of Bacillus subtilis succinate:menaquinone reductase is a protein of 202 residues containing two protoheme IX groups with bis-histidine axial ligation. Residues Kis13, His28, His70, His113, and His155 are the possible heme ligands. The transmembrane topology of this cytochrome was analyzed using fusions to alkaline phosphatase. The results support a proposed model with five transmembrane polypeptide segments and the N-terminus exposed to the cytoplasm. Mutant B. subtilis cytochromes containing a His13 --> Tyr, a His28 --> Tyr, and a His113 --> Tyr mutation, respectively, were produced in Escherichia coli, partially purified, and analyzed. In addition, succinate: menaquinone reductase containing the His13 --> Tyr mutation in the anchor subunit was overproduced in B. subtilis, purified, and characterized. The data demonstrate that His13 is not an axial heme ligand. Thermodynamic and spectroscopic properties of the cytochrome are, however, affected by the His13 --> mutation; compared to wild type, the redox potentials of both hemes are negatively shifted and the g(max) signal in the EPR spectrum of the high-potential heme is shifted from 3.68 to 3.50. From the combined results we conclude that His28 and His113 function as axial ligands to the low-potential heme, which is located in the membrane near the outer surface of the cytoplasmic membrane. Residues His70 and His155 ligate the high-potential heme, which is positioned close to His13 in the protein, near the inner surface of the membrane.
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| 3. |
- Hägerhäll, Cecilia, et al.
(författare)
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Two hemes in Bacillus subtilis succinate:menaquinone oxidoreductase (Complex II)
- 1992
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 31, s. 7411-7421
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Tidskriftsartikel (refereegranskat)abstract
- Succinate:menaquinone-7 oxidoreductase (complex II) of the Gram-positive bacterium B subtilis consists of equimolar amounts of three polypeptides; a 65-kDa FAD-containing polypeptide, a 28-kDa iron-sulfur cluster containing polypeptide, and a 23-kDa membrane-spanning cytochrome b558 polypeptide, The enzyme complex was overproduced 2-3-fold in membranes of B. subtilis cells containing the sdhCAB operon on a low copy number plasmid and was purified in the presence of detergent. The cytochrome b558 subunit alone was similarly overexpressed in a complex II deficient mutant and partially purified. Isolated complex II catalyzed the reduction of various quinones and also quinol oxidation. Both activities were efficiently albeit not completely blocked by 2-n-heptyl-4-hydroxyquinoline N-oxide. Chemical analysis demonstrated two protoheme IX per complex II. One heme component was found to have an E(m,7.4) of +65 mV and an EPR g(max) signal at 3.68, to be fully reducible by succinate, and showed a symmetrical alpha-band absorption peak at 555 nm at 77 K. The other heme component was found to have an E(m,7.4) of -95 mV and an EPR g(max) signal at 3.42, was not reducible by succinate under steady-state conditions, and showed in the reduced state an apparent split alpha-band absorption peak with maxima at 553 and 558 nm at 77 K. Potentiometric titrations of partially purified cytochrome b558 subunit demonstrated that the isolated cytochrome b558 also contains two hemes. Some of the properties, i.e., the alpha-band light absorption peak at 77 K, the line shapes of the EPR g(max) signals, and reactivity with carbon monoxide were observed to be different in B. subtilis cytochrome b558 isolated and in complex II. This suggests that the bound flavoprotein and iron-sulfur protein subunits protect or affect the heme environment in the assembled complex
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| 4. |
- Matsson, Mikael, et al.
(författare)
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The distal heme center in Bacillus subtilis succinate:menaquinone reductase is crucial for electron transfer to menaquinone
- 2000
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 39, s. 8617-8624
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Tidskriftsartikel (refereegranskat)abstract
- Succinate:quinone reductases are membrane-bound enzymes that catalyze electron transfer from succinate to quinone. Some enzymes in vivo reduce ubiquinone (exergonic reaction) whereas others reduce menaquinone (endergonic reaction). The succinate:menaquinone reductases all contain two heme groups in the membrane anchor of the enzyme: a proximal heme (heme b(P)) located close to the negative side of the membrane and a distal heme (heme b(D)) located close to the positive side of the membrane. Heme b(D) is a distinctive feature of the succinate:menaquinone reductases, but the role of this heme in electron transfer to quinone has not previously been analyzed. His28 and His113 are the axial ligands to heme b(D) in Bacillus subtilis succinate:menaquinone reductase. We have individually replaced these His residues with Leu and Met, respectively, resulting in assembled membrane- bound enzymes. The H28L mutant enzyme lacks succinate:quinone reductase activity probably due to a defective quinone binding site. The H113M mutant enzyme contains heme b(D) with raised midpoint potential and is impaired in electron transfer to menaquinone. Our combined experimental data show that the heme b(D) center, into which we include a quinone binding site, is crucial for succinate:menaquinone reductase activity. The results support a model in which menaquinone is reduced on the positive side of the membrane and the transmembrane electrochemical potential provides driving force for electron transfer from succinate via heme b(P) and heme b(D) to menaquinone.
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| 5. |
- Stenklo, Katarina, et al.
(författare)
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Chlorite dismutase from Ideonella dechloratans
- 2001
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Ingår i: Journal of Biological Inorganic Chemistry. - : Springer Science and Business Media LLC. - 0949-8257 .- 1432-1327. ; 6:5-6, s. 601-607
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Tidskriftsartikel (refereegranskat)abstract
- Chlorite dismutase has been purified from the chlorate-metabolizing bacterium Ideonella dechloratans. The purified enzyme is tetrameric, with a relative molecular mass of 25,000 for the subunit, and contains about 0.6 heme/subunit as isolated. Its catalytic properties are similar, but not identical, to those found for a similar enzyme purified earlier from the bacterium GR-1. The heme group in Ideonella chlorite dismutase is readily reduced by dithionite, in contrast to the GR-1 enzyme, and redox titration gave a value of -21 mV for the midpoint potential at pH 7. The heme group has been characterized by optical and EPR spectroscopy. It is high-spin ferric at neutral pH, with spectroscopic properties similar to those found for cytochrome c peroxidase. In the alkaline pH range, a low-spin compound is formed. A 22-residue N-terminal amino acid sequence has been determined and no homologue has been found in the protein sequence databases.
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